Article pubs.acs.org/molecularpharmaceutics Therapeutic Strategies for Gaucher Disease: Miglustat (NB-DNJ) as a Pharmacological Chaperone for Glucocerebrosidase and the Different Thermostability of Velaglucerase Alfa and Imiglucerase Olga Abian,*, ,!,§,% Pilar Alfonso,*, ,!,¥ Adrian Velazquez-Campoy,§,#,∇ Pilar Giraldo, ,!,¥,Ë Miguel Pocovi,!,¥,# and Javier Sancho§,# Unidad de Investigación Traslacional, Miguel Servet Universitary Hospital, Zaragoza, Spain !Aragon Health Sciences Institute (I+CS), Zaragoza, Spain §Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC)-Joint Unit, Spain %Centro de Investigación Biomedicá en Red en el Área Tematicá de Enfermedades Hepaticaś y Digestivas (CIBERehd) and ¥Centro de Investigación en Red de Enfermedades Raras (CIBERER), ISCIII, Spain #Department of Biochemistry and Cellular and Molecular Biology, University of Zaragoza, Spain ∇Fundación ARAID (ARAID-BIFI), Diputación General de Aragón, Spain ËService of Hematology, Miguel Servet Universitary Hospital, Zaragoza, Spain ABSTRACT: Gaucher disease (GD) is a disorder of glycosphingolipid metabolism caused by deficiency of lysosomal glucocerebrosidase (GlcCerase) activity, due to conformationally or functionally defective variants, resulting in progressive deposition of glycosylceramide in macrophages. The glucose analogue, N-butyldeoxynojirimycin (NB- DNJ, miglustat), is an inhibitor of the ceramide-specific glycosyltransfer- ase, which catalyzes the first step of glycosphingolipid biosynthesis and is currently approved for the oral treatment of type 1 GD. In a previous work, we found a GlcCerase activity increase in cell cultures in the presence of NB-DNJ, which could imply that this compound is not only a substrate reducer but also a pharmacological chaperone or inhibitor for GlcCerase degradation. In this work we compare imiglucerase (the enzyme currently used for replacement therapy) and velaglucerase alfa (a novel therapeutic enzyme form) in terms of conformational stability and enzymatic activity, as well as the effect of NB-DNJ on them. The interaction between these enzymes and NB-DNJ was studied by isothermal titration calorimetry. Our results reveal that, although velaglucerase alfa and imiglucerase exhibit very similar activity profiles, velaglucerase alfa shows higher in vitro thermal stability and is less prone to aggregation/precipitation, which could be advantageous for storage and clinical administration. In addition, we show that at neutral pH NB-DNJ binds to and enhances the stability of both enzymes, while at mildly acidic lysosomal conditions it does not bind to them. These results support the potential role of NB-DNJ as a pharmacological chaperone, susceptible of being part of pharmaceutical formulation or combination therapy for GD in the future. KEYWORDS: Gaucher disease, imiglucerase, velaglucerase alfa, NB-DNJ, calorimetry 1. INTRODUCTION they represent different degrees of severity along a broad Gaucher disease (GD, OMIM #230800) is an inherited lipid spectrum. The only two treatments currently approved for GD patients are enzyme replacement therapy (ERT) and substrate storage disorder caused by the insufficient activity of the 4 enzyme glucocerebrosidase (GlcCerase, EC 3.2.1.45),1 and it is reduction therapy (SRT). characterized by intralysosomal accumulation of glucocerebro- The first used therapeutic enzyme was alglucerase (Ceredase; side (glucosylceramide; GlcCer) that leads to dysfunction in Genzyme Corporation, Cambridge, MA), extracted from 2 human placental tissue.5 Due to major limitations of this multiple organ systems. Mutations in the glucocerebrosidase 6,7 gene generate misfolded or unstable, degradation-prone protein product, a recombinant form of the enzyme, imiglucerase variants with diminished catalytic activity or aberrant trafficking (Cerezyme; Genzyme Corporation, Cambridge, MA), was from the endoplasmic reticulum (ER) to the lysosome, reducing its lysosomal concentration and activity.3,4 Con- Received: June 22, 2011 sequently, GD is characterized by the presence of lipid-laden Revised: September 19, 2011 macrophages (Gaucher cells) in the liver, spleen, bone, and Accepted: October 11, 2011 lungs. Three types of GD have been described, but, actually, Published: October 11, 2011 © 2011 American Chemical Society 2390 dx.doi.org/10.1021/mp200313e |Mol. Pharmaceutics 2011, 8, 2390−2397 Molecular Pharmaceutics Article developed. A novel form of GlcCerase with an amino acid The X-ray structure of velaglucerase alfa is very similar to sequence identical to that of the natural human protein, those of recombinant GlcCerases produced in other expression recently approved in the USA for the treatment of Gaucher systems, with the R495H mutation in imiglucerase having no disease, is gene-activated human GlcCerase (velaglucerase alfa, effect on the secondary structure. The main difference between VPRIV, Shire HGT, Dublin, Ireland).8 Both enzymes act like imiglucerase and velaglucerase alfa concerns their glycan their natural active equivalent GlcCerase to break down the structures, with the latter containing longer chain high- GlcCer that has accumulated in Gaucher cells.9,10 ERT mannose type glycans compared to core mannose structures improves the visceral and hematologic manifestations of the found on imiglucerase. This difference in their glycosylation disease,11,12 without serious adverse effects.9,13 Disadvantages patterns appears to be related to the increased cellular uptake of of ERT include regular intravenous infusions, little direct effect velaglucerase alfa over imiglucerase, which could lead to a faster on the neurological manifestations of the central nervous response and improvement of clinical parameters in patients system because of its inability to cross the blood−brain and, potentially, to increased therapeutic efficacy.46 barrier,4,10,14 and high cost precluding many patients from Different studies suggest that a significant amount of the access to therapy.15 therapeutically administered GlcCerase may become inactive The second therapeutic approach is SRT with N-butyldeox- due to unfolding and/or lack of structural stability during its ynojirimycin (NB-DNJ) (miglustat; Zavesca; Actelion Pharma- transit through plasma, because of body temperature and the ceuticals, South San Francisco, CA, USA), an iminosugar that neutral pH of blood before reaching the target lysosomal reversibly inhibits glucosylceramide synthase and reduces membrane.48,49 GlcCer biosynthesis.16,17 Studies showed that this oral In this work we have characterized effects of pH and NB- treatment improved organ volumes, bone density and DNJ binding on the thermostability and the activities of hematological parameters of patients with mild to moderate imiglucerase and velaglucerase alfa in an attempt to understand type 1 Gaucher.18−23 Other possible treatment strategies the physical basis of its potential chaperoning role. Both include the use of pharmacological chaperones, which are enzymes have been assayed at two pH values: neutral pH, small molecules designed specifically to bind to their target reflecting the endoplasmic reticulum environment (for macromolecule (e.g., a mutated enzyme) and, ideally, may endogenous GlcCerase) and the blood environment (for induce refolding and conformational stabilization24−27 against ERT GlcCerase), and mildly acidic pH, reflecting the lysosomal unfolding and/or protection against degradation by the ER- environment. associated quality-control system.28−30 Glycoside hydrolases are exposed to different intracellular environments where their 2. EXPERIMENTAL SECTION function must be finely tuned: they are involved in the turnover 2.1. Enzymes and Chemicals. Lyophilized imiglucerase of intracellular substrates in the acidic environment (pH < 5.2) (Cerezyme; Genzyme Corporation, Cambridge, MA) and 31 of the lysosome; however, these enzymes are synthesized and velaglucerase alfa (VPRIV; Shire HGT, Dublin, Ireland) were folded at the neutral-pH environment of the ER, exported to reconstituted in the appropriate buffer and dialyzed for 6−16 h the Golgi apparatus for sorting, and subsequently trafficked to (10 kDa molecular mass cutoff) and degassed prior to the lysosomes. corresponding experiment. NB-DNJ (Zavesca, Actelion Phar- It has been demonstrated that sugar-analogue reversible maceuticals, South San Francisco, CA, USA) was dissolved in inhibitors can increase the activity of mutant lysosomal the same buffers employed for the enzymes. enzymes in patient fibroblasts by acting as pharmacological 2.2. Differential Scanning Calorimetry (DSC). The heat chaperones (chaperone mediated therapy).32−37 It has also capacity of proteins was measured as a function of temperature been reported that the preincubation of GlcCerase with with a high-sensitivity differential scanning VP-DSC micro- isofagomine (IFG), a slow-binding inhibitor, effectively calorimeter (MicroCal, Northampton, MA). Protein samples stabilizes GlcCerase in vitro and appears to protect the enzyme and reference solutions were properly degassed and carefully against thermal and chemical denaturation.35,38−41 loaded into the cells to avoid bubble formation. Thermal The mature GlcCerase enzyme has 497 amino acid residues denaturation scans were performed with freshly prepared with a molecular weight of <62 kDa.42,43 The recombinant buffer-exchanged protein solutions. The baseline of the enzyme imiglucerase differs from human GlcCerase in a single instrument was routinely recorded